sign in sign up
<<
Home , Cyanate, Sodium cyanate

Application Note 200

Direct Determination of in a Solution and a Urea-Containing Protein Buffer

INTRODUCTION Urea is commonly used in protein purification, detection.9 This method was used to evaluate including large scale purification of recombinant proteins the efficiency of cyanate scavengers and make for commercial purposes, and in recombinant protein recommendations for protein buffers that reduced manufacturing to denature and solubilize proteins.1–3 Some cyanate accumulation and subsequent carbamylation proteins are readily soluble and denature at moderate reactions. The authors evaluated separate 0.1 M citrate, urea concentrations (4–6 M), however, most solubilize phosphate, and borate buffers in 8 M urea across a and denature at higher concentrations (8–10 M).4–6 Urea pH range of 5–9. They reported that the citrate (pH = 6) degrades to cyanate and ammonium in aqueous solutions. buffered urea solution demonstrated the best suppression The maximum rate of cyanate production occurs near of cyanate accumulation (<0.2 mM cyanate). However, neutral pH, the typical pH range of biological buffers.7 phosphate buffers at pH 6 and 7 (<0.5 mM cyanate) Cyanate can carbamylate proteins through a reaction were preferred over the citrate buffers because citrate with free amino, carboxyl, sulfhydryl, imidazole, phenolic actively carboxylates proteins. (It is sometimes used as hydroxyl, and phosphate groups.8 These are unwanted a carboxylating agent.) While this analytical method modifications that can alter the protein’s stability, was effective, the authors believed it could be improved function, and efficiency. While some of these reactions by using a high-capacity hydroxide-selective anion- can be reversed by altering the pH of the solution, exchange column with better chloride-cyanate resolution. cyanate-induced carbamylation reactions to N-terminal Hydroxide eluent delivers better sensitivity than amino acids, however–such as arginine and lysine–are /. Improved resolution between irreversible.8 Urea solutions are commonly deionized chloride and cyanate, combined with the capability to remove cyanate for this reason. Unfortunately, high to inject more concentrated samples due to the higher cyanate concentrations can accumulate in urea solutions column capacity also provides increased method regardless of prior deionization, with some urea buffers sensitivity. reporting cyanate concentrations as high as 20 mM.7 This application note shows determination of cyanate An accurate, sensitive method for measuring cyanate in in samples of 8 M urea, 8 M urea with 1 M chloride, and high ionic strength matrices is needed to help monitor and 8 M urea with 1 M chloride and 50 mM phosphate buffer control quality in these buffers. (pH = 8.4) using a Reagent-Free™ Chromatography Ion chromatography (IC) is an ideal method for (RFIC™) system. This method provides improved cyanate determination. A 2004 publication showed sensitivity, allows smaller volume sample injections, determination of cyanate in urea solutions by IC using a lowers the required dilution, and demonstrates higher ® resolution of cyanate from chloride compared to the Dionex IonPac AS14 column with 3.5 mM Na2CO3/ previously published method. Cyanate is separated using 1.0 mM NaHCO3 eluent, and suppressed conductivity a hydroxide-selective IonPac AS15 (5-µm) column with chloride (NaCl) (VWR, JT3624) electrolytically generated 25 mM potassium hydroxide (NaOCN) (Aldrich, 185086) eluent and suppressed conductivity detection. The Sodium phosphate, dibasic anhydrous (Na2HPO4) IonPac AS15 column is a 3 x 150 mm high-capacity (VWR, JT3828) (60 µEq/column) column, with a smaller particle size, Sodium phosphate, monobasic monohydrate diameter, and length than the IonPac AS14 column (NaH2PO4∙H2O) (VWR, JT3818) described in a previous method. These changes improve Urea (H2NCONH2) (VWR, JT4204-1) sensitivity, reduce eluent consumption, and allow higher sample throughput. The mobile phase is electrolytically Urea Matrix Sample Solutions generated, which reduces labor, improves consistency, and Urea solutions were prepared from solid compounds provides the reproducibility of an RFIC system. Linearity, in 18.2 MΩ-cm deionized water and diluted 50× prior to limit of detection (LOD), precision, recoveries, and cyanate determinations: stability of cyanate in urea as a function of temperature 8 M urea are discussed. 8 M urea with 1 M chloride 8 M urea with 1 M chloride and 50 mM phosphate EXPERIMENTAL (pH = 8.4) Equipment All solutions containing urea were prepared the same Dionex ICS-3000 RFIC-EG™ system consisting of: day of the experiments unless otherwise stated. SP Single gradient pump DC Detector and Chromatography module, single or CONDITIONS dual temperature zone configuration Columns: IonPac AS15 5-µm Analytical CD Conductivity Detector (3 x 150 mm, P/N 057594) EG Eluent Generator IonPac AG15 5-µm Guard, AS Autosampler with Sample Tray Temperature (3 x 30 mm, P/N 057597) Controlling option and 1.5 mL sample tray Eluenta: 25 mM KOH EluGen® EGC II KOH cartridge (P/N 058900) Eluent Source: EGC II KOH with CR-ATC Continuously Regenerated Anion Trap Column Flow Rate: 0.5 mL/min (CR-ATC, P/N 060477) Column Temperature: 30 ºC Chromeleon® 6.8 Chromatography Workstation Tray Temperature: 4 ºCb Virtual Column™ Separation Simulator (optional) Injection Volume: 5 µL Sample Vial kit, 0.3-mL polypropylene with caps and Detection: Suppressed conductivity, septa (P/N 055428) ASRS® 300 (2 mm, P/N a This application can be performed on other Dionex 064555), recycle mode, 31 mA RFIC-EG systems. Background Conductivity: <1 µS REAGENTS AND STANDARDS Baseline Noise: <2 nS Reagents System Backpressure: ~2200 psi Deionized water, Type 1 reagent-grade, 18.2 MΩ-cm Run Time: 22 min resistivity, freshly degassed by ultrasonic agitation and a Add a step change at 12 min to 65 mM KOH when applied vacuum. determining cyanate in samples containing phosphate. Use only ACS reagent grade chemicals for all The eluent conditions are: 25 mM KOH for 0 to 12 min, reagents and standards. and 65 mM KOH from 12 to 20 min, recycle mode, 81 mA. Chloride standard (1000 mg/L; Dionex, P/N 037159) b Temperature was maintained at 4 °C using the , anhydrous (Na2CO3) (VWR, JT3602) temperature-controlled autosampler tray to minimize changes in cyanate concentration.

2 Direct Determination of Cyanate in a Urea Solution and a Urea-Containing Protein Buffer Using a Reagent-Free Ion Chromatography System PREPARATION OF SOLUTIONS AND REAGENTS Prepare the combined 8 M urea, 1 M It is essential to use high quality, Type 1 water, with (NaCl, FW 58.44 g/mole) matrix sample solution in a resistivity of 18.2 MΩ-cm or greater, and it must be a similar manner using 48.05 g urea, 5.84 g sodium relatively free of dissolved dioxide. Degas the chloride, and deionized water in a 100 mL volumetric deionized water using ultrasonic agitation with applied flask. vacuum. Prepare the combined 8 M urea, 1 M sodium chloride, and 50 mM phosphate (49.5 mM sodium

1 M Stock Cyanate Standard Solution phosphate monobasic (NaH2PO4∙H2O), 0.5 mM sodium

To prepare a 1 M stock cyanate standard solution, phosphate dibasic (Na2HPO4) matrix solution using dissolve 6.50 g of sodium cyanate (NaOCN, FW 65.01 g/mol) 48.05 g urea, 5.84 g sodium chloride, 0.683 g sodium with deionized water in a 100 mL volumetric flask and bring to phosphate monobasic (NaH2PO4∙H2O, FW 58.44 g/mole), volume. Gently shake the flask to thoroughly mix the solution. 0.007 g sodium phosphate dibasic (Na2HPO4, FW 141.96 g/mole), and deionized water in a 100 mL Primary and Working Cyanate Standard Solutions volumetric flask. Bring to volume, and mix thoroughly To prepare a 1.00 mM cyanate primary standard, (pH = 8.4). pipette 100 µL of the 1 M cyanate stock standard solution To prepare 100-, 50-, and 10-fold dilutions of the into a 100-mL volumetric flask, bring to volume with matrix sample solutions for the dilution experiments, deionized water, and shake the flask gently to mix. pipette 200, 400, and 2000 µL, respectively, of the matrix To prepare 1, 2, 4, 10, 20, 50, and 100 µM cyanate sample solution into a (tared) 20 mL polypropylene individual working standards, pipette 50, 100, 200, 500, scintillation vial and add deionized water until a total 1000, 2500, and 5000 µL, respectively of the 1.00 mM cyanate weight of 20.00 g is reached. primary standard solution into separate 50 mL volumetric flasks. Bring to volume with deionized water and shake PRECAUTIONS gently to mix. The urea solutions must be stored frozen at -20 or Prepare the 0.13, 0.25, and 0.50 µM cyanate detection -40 °C and defrosted before use or prepared fresh daily limit standards by diluting the 1.0 µM cyanate working as the cyanate concentrations increase over time and with standard using serial dilutions. For example, a portion of increased temperature. the 1.0 µM cyanate standard is diluted 50% to 0.50 µM cyanate. The 0.13 and 0.25 µM cyanate standards are SYSTEM SETUP prepared in a similar way from the 0.25 and 0.50 µM Refer to the instructions in the ICS-3000 Installation10 cyanate standards, respectively. and Operator’s11 manuals, AS Autosampler Operator’s Store all standards at 4 °C. Prepare the 1–20 µM manual,12 and column,13 and suppressor14 product manuals standard solutions weekly and the primary and stock for system setup and configuration. standard solutions monthly.

Urea Matrix Sample Solutions

To prepare 8 M urea (H2NCONH2, FW 60.06 g/mole) matrix sample solution, dissolve 48.05 g urea with deionized water in a 100 mL volumetric flask, dilute to volume, and mix thoroughly.

Application Note 200 3 RESULTS AND DISCUSSION Column: I onPac® AG15 5-µm, Eluent Source: EGC II KOH Method Development and Optimization AS15 5-µm (3 mm) Temperature: 30 °C Eluent: 25 mM KOH Flow Rate: 0.50 mL/min The challenge in this application is to accurately Inj. Vol: 5 µL determine low concentrations of cyanate in a matrix with 0.58 Detection: ASRS® 300, recycle, 31 mA Sample Prep.: 50-fold dilution high concentrations of salts (up to 20,000× higher) and to 5 Peaks: 1-2. Unknown — µM resolve a cyanate peak eluting near a significantly larger 3. Chloride — chloride peak. In a previous study, cyanate was separated 4. Cyanate 2.3 5. Carbonate — on a Dionex IonPac AS14 column using carbonate/ µS bicarbonate eluent at a flow rate of 1.2 mL/min, with 9 3 suppressed conductivity detection. The authors reported 4 a 1.3 min retention time difference between chloride and 12 cyanate. However, cyanate is not fully resolved from 0.41 chloride in urea solutions containing 1 M chloride. The 0 510 15 2220 Minutes authors also reported that the chromatogram’s baseline 25298 was affected when 8 M urea solutions were injected. Figure 1. Determination of 2 µM cyanate by ion chromatography. As urea eluted through the column, the baseline of the chromatogram increased >2 µS at approximately 3 min, To refine the results obtained by Virtual Column then slowly drifted downward to the original baseline. simulator, the retention times of a 5 µL injection of 1 mM The cyanate detection limit corresponding to the undiluted cyanate, chloride, and carbonate were determined on an urea solution is high (200 µM). An RFIC system with a IonPac AS15 5-µm, 3 x 150 mm column, using hydroxide-selective column and electrolytically generated 20, 25, and 30 mM potassium hydroxide at 0.5 mL/ hydroxide eluent can be easily optimized to resolve min and 30 °C. The experiments showed cyanate well low concentrations of cyanate from high concentrations resolved from chloride and carbonate by 3.2 min (Rs > of chloride in urea samples. Electrolytically generated 3) using either 20 or 25 mM potassium hydroxide, and, hydroxide eluent improves sensitivity and reduces as expected, retention times decreased with increasing baseline noise. eluent strength. 25 mM potassium hydroxide eluent was In order to optimize conditions and to minimize selected for this assay. Figure 1 demonstrates good peak the time required to select the hydroxide-selective, response and peak asymmetry for 2 µM cyanate separated high-capacity column with the highest resolution for on the IonPac AS15 5-µm column using electrolytically cyanate, the authors used the Chromeleon Virtual Column generated 25 mM potassium hydroxide at 0.5 mL/min. simulator to model separation conditions. Nitrite was During the initial method evaluation, the effects used to model cyanate because it is not currently part of column overload using 8 M urea samples with and of the Virtual Column database and was chosen after without spiked concentrations of 1 M sodium chloride reviewing chromatograms in column manuals for various were tested. The chromatograms of undiluted 8 M urea high-capacity hydroxide-selective columns. (Only showed a similar baseline disturbance (3 µS) from urea as these types of columns were considered because some reported in the literature9 (not shown). As urea elutes from urea-containing solutions contain molar concentrations the column, the baseline shifts upward at approximately of chloride and other anions that may overload low- 3 min then slowly back to the original baseline at 15 min. capacity columns). Virtual Column simulator was used to All tested urea-containing solutions demonstrated similar evaluate the separation of chloride, nitrite, carbonate, and results. The magnitude of the baseline shifts reduced with phosphate with isocratic hydroxide eluents at dilution (Figures 2–4). Urea contains minor unidentified 30 °C. The simulator results demonstrated that the IonPac that do not interfere with the cyanate peak and elute AS15 column using 20 mM KOH provided the optimum from the column in less than 20 min. 8 M urea samples chloride/nitrite and nitrite/carbonate resolution with did not overload the column. To determine whether the

Rs > 3. The Virtual Column simulator proved to be a molar concentrations of chloride expected in the samples valuable tool in helping accelerate method development would cause column overloading, 20-, 50-, and 100-fold by eliminating the time required to select a column and dilutions of 8 M urea solutions containing 1 M chloride eluent suitable for this application. were injected. The chromatography of the 20-fold dilution

4 Direct Determination of Cyanate in a Urea Solution and a Urea-Containing Protein Buffer Using a Reagent-Free Ion Chromatography System samples (50 mM chloride) showed distorted chloride volume than the AS14 column. These differences provide peaks typical of column overloading. At least 50× improvement in peak response and sensitivity. This allows dilutions of 8 M urea with 1 M chloride are required to for a smaller sample (5 µL) to be injected, resulting in avoid this phenomenon and to obtain adequate resolution less column overload and longer column life. The limit of of chloride and cyanate. quantification, using 10× S/N, is 0.8 µM (1.0 µM, S/N 11.6). Method Qualification To qualify the cyanate method, linearity, system Determination of Cyanate in Urea and Urea-Containing noise, limit of quantification, and LOD were evaluated. Solutions The peak area response of cyanate was determined from The method was applied to 50-fold dilutions of 1 to 100 µM, using triplicate injections of the calibration 8 M urea, 8 M urea with 1 M chloride, and 8 M urea with standards. The linearity of cyanate peak area responses 1 M chloride and 50 mM phosphate buffer (pH 8.4). The was determined with Chromeleon software using a least authors determined the urea and urea-containing solutions squares regression fit. The resulting correlation coefficient had similar cyanate concentrations of 1.1 ± 0.1 µM in (r2) was 0.9993. 50× diluted solutions (Table 1). The cyanate peak was The peak-to-peak baseline noise was measured in well resolved from chloride and carbonate, and had good 1 min segments from 20 to 60 min without injecting a peak shape (Figure 2A). An acceptably small baseline sample. The noise was acceptably low (0.95 ± 0.13 nS drift (<0.15 µS) from urea was observed starting at (n = 3)). To determine the limit of detection, seven 2.4 min and ending at about 15 min. In urea-containing replicates of 0.13, 0.25, and 0.50 µM cyanate were solutions with molar concentrations of chloride, cyanate injected. The peak responses of cyanate were compared elutes on the base of the large chloride peak and is against the baseline noise using 3× S/N. The LOD was not fully resolved from chloride (Figure 3A). The 0.25 µM cyanate (S/N 3.01). These detection limits are chromatography is similar for urea-containing solutions significantly lower than previously reported (2 µM) using with chloride and phosphate (Figure 4A). an IonPac AS14 column with bicarbonate/carbonate To determine the method accuracy, multiple additions eluents.9 This improvement is likely due to the advantages of cyanate (1.2, 2.2, and 3.6 µM) were added to 50× of using electrolytically generated hydroxide eluent with dilutions of 8 M urea samples. In addition, 50× dilutions suppressed conductivity detection. The IonPac AS15 of 8 M urea with 1M chloride and 8 M urea with 1 M column has similar capacity per column but 30% less chloride, and 50 mM phosphate buffer (pH = 8.4) were

Table 1. Recoveries of Cyanate in Urea Solutions (Dilution Factor: 50×) Matrixa Amount RSD Amount Amount RSD Recovery Amount Amount RSD Recovery Amount Amount RSD Recovery Present Added Measured (%) Added Measured (%) Added Measured (%) (µM) (µM) (µM) (µM) (µM) (µM) (µM) A 1.13 0.50 1.20 2.26 0.64 96.7 2.20 3.32 1.38 99.7 3.62 4.92 0.87 103.6 B 1.11 1.07 0.91 1.88 1.03 93.1 1.31 2.43 1.44 100.4 2.22 3.48 0.55 104.4 C 1.00 1.41 0.81 1.60 1.10 89.5 1.24 2.27 0.69 101.1 1.70 2.66 1.16 98.0 A) 8 M Urea B) 8 M Urea, 1 M Chloride C) 8 M Urea, 1 M Chloride, 50 mM Phosphate Buffer pH = 8.4

Application Note 200 5 similarly spiked with 0.9–2.2 µM, and 0.8–1.7 µM Column: IonPac® AG15 5-µm, Temperature: 30 °C cyanate, respectively. The calculated recoveries were AS15 5-µm (3 mm) Flow Rate: 0.50 mL/min Eluent: 25 mM KOH Inj. Vol: 5 µL >89% for all solutions (Table 1). The chromatograms of Eluent Source: EGC II KOH Detection: ASRS® 300, recycle, 31 mA the 50× dilution of 8 M urea and the same solution spiked Sample Prep.: 50-fold dilution 0.85 Samples: A. 8 M urea with 3.6 µM cyanate are shown in Figure 2. Peaks 6–8 are B. Sample A with 2.2 µM of added cyanate unidentified ions from the urea matrix that elute from the column within 20 min, while run time was extended to 22 A B Peaks: 1-2. Unknown — — µM min to ensure that these ions were fully eluted before the µS 3. Chloride — — 5 4. Cyanate 1.1 3.1 next injection. The chromatograms of unspiked and spiked 3 5. Carbonate — — 6-8. Unknown — — with 2.2 µM cyanate in 50-fold dilution of 8 M urea 1 M 4 2 chloride are shown in Figure 3. The chromatograms of 1 B 8 M urea with 1 M chloride and 50 mM phosphate buffer A 0.35 (pH=8.4), both unspiked and spiked with 1.7 µM cyanate 0 510 15 2220 Minutes and diluted 50× are shown in Figure 4. 25299 To determine the retention time and peak area Figure 2. Comparison of A) 8 M urea B) 8 M urea with precisions seven replicate injections of 2 µM cyanate were 2.2 µM cyanate. spiked into deionized water, 50× dilutions of 8M urea, 8 M urea with 1M chloride, and 8 M urea with 1M chloride and 50 mM phosphate buffer (pH 8.4). Column: IonPac® AG15 5-µm, Flow Rate: 0.50 mL/min Cyanate had similar retention times for all samples— AS15 5-µm (3 mm) Inj. Vol: 5 µL Eluent: 25 mM KOH Detection: ASRS® 300, recycle, 31 mA 8.99 to 9.07 min (Table 2). The retention time and peak Eluent Source: EGC II KOH Sample Prep.: 50-fold dilution area precisions were <0.1 and <2 % for all three samples. Temperature: 30 °C Samples: A. 8 M urea, 1 M chloride B. Sample A with 2.2 µM of added cyanate 0.58

Sample Stability 3 A B The accumulation of cyanate in urea as a function of Peaks: 1-2. Unknown — — µM 3. Chloride 20,000 20,000 temperature is frequently discussed in the literature.1,4–9 4. Cyanate 1.1 3.5 µS 5. Carbonate — — 5 6-8. Unknown — —

4 Table 2. Retention Time and Peak Area 2 1 6 Precisions of 2 µM Cyanate Spiked Into B 7 8 50-fold Dilution of Urea Solution A 0.35 Matrixa Retention Time RSD Peak Area 0 510 15 2220 Minutes (min) RSD 25300 Deionized Water 9.07 0.02 1.06 Figure 3. Comparison of A) 8 M urea 1 M chloride B) 8 M urea 1 M chloride with 2.2 µM cyanate. A 9.07 0.02 1.65 B 9.03 0.04 0.65 C 8.99 0.06 1.69 To determine the stability of cyanate in urea over 4 days, cyanate concentrations were determined from 50-fold A) 8 M Urea B) 8 M Urea, 1 M Chloride dilutions of 8 M urea, 8 M urea with 1 M chloride, and C) 8 M Urea, 1 M Chloride, 50 mM Phosphate Buffer pH = 8.4 8 M urea with 1 M chloride and 50 mM phosphate buffer n = 7 solutions (pH = 8.4). The solutions were stored at a Freshly prepared solutions -40 °C, 4 °C (AS autosampler tray), and 25 °C during the four-day experiment. To elute phosphate in the phosphate buffered urea solution, the method was modified with a step change to 65 mM KOH after the carbonate peak at 12 min.

6 Direct Determination of Cyanate in a Urea Solution and a Urea-Containing Protein Buffer Using a Reagent-Free Ion Chromatography System CONCLUSION Column: IonPac® AG15 5-µm, Eluent Source: EGC II KOH AS15 5-µm (3 mm) Temperature: 30 °C Urea degrades to cyanate, an unwanted contaminant Eluent: 25 mM KOH for 12 min, Flow Rate: 0.50 mL/min step change to 65 mM KOH, Inj. Vol: 5 µL in urea-containing buffers used for protein purification. 65 mM KOH from 12 to 20 min Detection: ASRS® 300, recycle, 31 mA Using a high-capacity anion-exchange column with Sample Prep.: 50-fold dilution Samples: A. 8 M urea, 1 M chloride suppressed conductivity detection, the authors accurately B. Sample A with 2.2 µM of determined low (µM) concentrations of cyanate in 50-fold 3 7 added cyanate 0.85 dilutions of 8 M urea and urea solutions containing A B Peaks: 1-2. Unknown — — µM molar concentrations of chloride and mM concentrations 3. Chloride 20,000 20,000 4. Cyanate 1.0 2.3 of phosphate. This method allows fast, accurate 5. Carbonate — — µS determination of cyanate in urea-containing solutions. A 6. Unknown — — 5 7. Phosphate 1,000 1,000 Reagent-Free IC system ensures the highest precision, 2 4 6 eliminates the need to prepare eluents, and eliminates 1 B possible eluent preparation errors. A 0.35 0 510 15 20 Minutes 25301 REFERENCES Figure 4. Comparison of A) 8 M urea 1 M chloride and 50 mM 1. Holtham, S.B.; Schütz, F. The effect of cyanate on the phosphate buffer (pH = 8.4) to B) Sample A spiked with 1.2 µM stability of proteins. Biochim. Biophys. Acta, 1949, 3, cyanate. 65–81. 2. Hoylaerts, M.; Chuchana, P.; Verdonck, P.; Roelants, P.; Weyens, A.; Loriau, R.; De Wilde, M.; Bollen, A. Large scale purification and molecular 8 M Urea, Defrosted from –40 °C characterization of human recombinant 1-proteinase 8 M Urea, at 25 °C 8 M Urea, at 4 °C inhibitor produced in yeasts. J. Biotechol., 1987, 5, 1000 181–197. 900 800 3. Amersham Pharmacia Biotech. The Recombinant 700 600 Protein Handbook. Protein amplification and simple 500

Cyanate (µM) 400 purification. Edition AA, 18-1142-75. 4-7 Amersham 300 200 100 Pharmacia Biotech, Piscataway, NJ. 2000. 4–7. 0 0102030405060708090 100 4. Dirnhuber, P.; Schütz, F. The isomeric transformation h 25302 of urea into ammonium cyanate in aqueous solutions. J. Biochem., 1948, 42, 628–632. Figure 5. Effect of temperature on cyanate from urea solutions. 5. Marier, J.R.; Rose, D. Determination of cyanate, and a study of its accumulation in aqueous solutions of urea. Anal. Biochem., 1964, 7, 304–314. The experiments showed the total cyanate 6. Reh, G.; Spelzini, D.; Tubio, G.; Pico, G.; Farruggia, concentrations were stable in 8 M urea when stored at B. Partition features and renaturation enhancement -40 °C. However, cyanate concentration increased more of chymosin in aqueous two-phase systems. J. than 10-fold over four days when stored at 4 °C (from Chromatogr. B., 2007, 860, 98–105. 6 to 75 µM) and increased significantly when stored at 7. Hagel, P.; Gerding, J.J.T.; Fieggen, W.; Bloemendal, 25 °C (from 24 to 886 µM) (Figure 5). Cyanate had H. Cyanate formation in solutions of urea. I. similar stability in 8 M urea with 1 M chloride and Calculation of cyanate concentrations at different 8 M urea with 1 M chloride and 50 mM phosphate buffer temperature and pH. Biochim. Biophys. Acta, 1971, as in the 8 M urea solutions. Cyanate accumulation in 243, 366–373. urea was not inhibited by the phosphate buffer. In the 8. Bennion, B.J.; Daggett, V. The molecular basis previous study, the authors reported that scavengers–in for the chemical denaturation of proteins by urea. addition to phosphate and other buffers–were needed to Proceedings of National Academy of Science (PNAS), effectively suppress the accumulation of cyanate in urea.9 2003, 100, 5142–5147. These results agree with the previous study.

Application Note 200 7 9. Lin, M-F.; Williams, C.; Murray, M.V.; Conn, G.; SUPPLIERS Ropp, P.A. Ion chromatographic quantification of Fisher Scientific International Inc., Liberty Lane, cyanate in urea solutions: estimation of the efficiency Hampton, NH 03842 Tel: 1-800-766-7000 of cyanate scavengers for use in recombinant protein www.fisherscientific.com manufacturing. J. Chromatogr. B., 2004, 803(2), VWR International, Inc., Goshen Corporate Park West, 353–362. 1310 Goshen Parkway, West Chester, PA 19380 10. Dionex Corporation. Installation Instructions for Tel: 1-800-932-5000 www.vwrsp.com ICS-3000 Ion Chromatography System; Document Sigma-Aldrich, Inc., P.O. Box 951524, Dallas, TX Number 065032. Dionex Corporation, Sunnyvale, 75395-1524 Tel: 1-800-325-3010 CA. 2005. www.sigmaaldrich.com 11. Dionex Corporation. Operator’s Manual for ICS-3000 Ion Chromatography System; Document Number 065031. Dionex Corporation, Sunnyvale, CA. 2005. 12. Dionex Corporation. Operator’s Manual for AS Autosampler; Document Number 065051. Dionex Corporation, Sunnyvale, CA. 2005. 13. Product Manual for IonPac AG15 Guard and AS15 Analytical Columns; Document Number 031362. Dionex Corporation, Sunnyvale, CA. 2002. 14. Product Manual for the Anion Self-Regenerating Suppressor 300 and Cation Self-Regenerating Suppressor 300; Document Number 031956. Dionex Corporation, Sunnyvale, CA. 2007.

EluGen, IonPac, ASRS, and Chromeleon are registered trademarks and Virtual Column, Reagent-Free, RFIC, and RFIC-EG are trademarks of Dionex Corporation

Passion. Power. Productivity.

Dionex Corporation North America Europe Asia Pacific 1228 Titan Way U.S. (847) 295-7500 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735 P.O. Box 3603 Canada (905) 844-9650 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190 Sunnyvale, CA Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Switzerland (41) 62 205 9966 Taiwan (886) 2 8751 6655 South America 94088-3603 United Kingdom (44) 1276 691722 LPN 2034 PDF 08/16 Brazil (55) 11 3731 5140 © Dionex Corporation (408) 737-0700 www.dionex.com 2016